干扰mTOR对HHV-8感染的神经细胞SH-SY5Y的增殖和迁移能力的影响

Effects of Interfering mTOR on Proliferation and Migration of HHV-8-Infected Nerve Cells SH-SY5Y

目的研究干扰mTOR对人类疱疹病毒8(HHV-8)感染的神经细胞SH-SY5Y的增殖和迁移能力的影响。方法通过Western blot实验和qRT-PCR法验证HHV-8感染的SH-SY5Y神经细胞的mTOR表达水平。采用Lipofectamine 2000分别将si-mTOR与si-NC转染进KSHV感染的SH-SY5Y细胞中,采用Western blot和qRT-PCR技术检测转染效率;qRT-PCR检测HHV-8病毒相关因子LANA、RTA、K8.1的mRNA表达水平;采用CCK-8法和划痕实验检测细胞增殖和迁移能力;利用PI染色结合流式细胞术检测细胞周期;采用Western blot检测周期相关蛋白cyclin D1和p27的表达。结果Western blot和qRT-PCR结果显示HHV-8感染的SH-SY5Y细胞中mTOR的表达水平高于SH-SY5Y细胞(P<0.05)。在干扰mTOR的HHV-8感染的SH-SY5Y细胞中,HHV-8病毒相关因子LANA、RTA、K8.1的表达水平均降低(P<0.05)。CCK-8和划痕实验结果表明干扰mTOR组细胞增殖、迁移能力低于对照组(P<0.05)。流式细胞术结果提示干扰mTOR组的细胞周期阻滞在G0/G1期(P<0.05)。Western blot结果显示cyclin D1表达水平降低,p27表达水平升高(P<0.05)。结论干扰mTOR能够降低HHV-8感染的SH-SY5Y细胞的增殖和迁移能力,并将其细胞周期阻滞在G0/G1期,针对mTOR的靶向调控在治疗HHV-8感染引起的神经系统疾病有一定的作用。

Objective To study the effect of interfering mTOR on the proliferation and migration of HHV-8 infected nerve cells SH-SY5 Y.Methods Western blot and qRT-PCR were used to detect the level of mTOR in HHV-8 infected and uninfected SH-SY5 Y cells.Lipofectamine 2000 was used to transfect si-mTOR and si-NC into HHV-8 infected SH-SY5 Y cells respectively.Western blot and qRT-PCR were used to detect the transfection efficiency.The mRNA expression levels of HHV-8 virus-related factors LANA,RTA and K8.1 were detected by qRT-PCR.CCK-8 assay and scratch assay were used to detect cell proliferation and migration.PI staining and flow cytometry were used to detect the cell cycle.The expression levels of cyclin D1 and p27 were detected by Western blot.Results The expression level of mTOR in HHV-8 infected SH-SY5 Y cells was higher than that in SH-SY5 Y cells(P<0.05).After treatment with si-mTOR,the expression levels of LANA,RTA and K8.1 were all decreased(P<0.05).Cell proliferation and migration ability in mTOR interfering group were lower than those in control group(P<0.05).Flow cytometry results indicated that the cell cycle was arrested at the G0/G1 phase in the mTOR interfering group(P<0.05).Western blot results showed that the expression of cyclin D1 was decreased and the expression of p27 was increased(P<0.05).Conclusion Interfering mTOR can reduce the proliferation and migration ability of HHV-8 infected SH-SY5 Y cells,and block the cell cycle at the G0/G1 phase.The targeted regulation of mTOR may plays a certain role in the treatment of nervous system diseases caused by HHV-8 infection.